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Effect of trypsin inhibitor on islet isolation from fresh and cold preserved rat pancreas
Effect of Trypsin Inhibitor on Islet Isolation From Fresh and Cold Preserved Rat Pancreas W.-T. Lu, J.R.T. Lakey, J.-H. Juang, B.R.-S. Hsu, and R.V. Rajotte
O
BTAINING more islets has limited the progress of human islet transplantation. Multiple factors, including ischemia time, different types of collagenase, distension degree applied to the pancreas, mechanic trauma, and variable digestion might be involved in the low islet recovery rate from a cadaveric pancreas.1–3 Among these factors, the collagenase digestion phase is thought to be the most crucial issue because of the between batch variation.4 In addition to the variable content of exogenous proteases in different collagenase blends, it is more attractive to investigate the variable activity of endogenous proteases released by pancreatic acinar cells during different digestion conditions.5 Wolters et al, described a positive effect (⫹48 %) of a collagenase solution supplemented with 10% bovine serum albumin as a protease inhibitor to increase rat islet yield.6 Two studies reported that pefabloc reduces trypsin activity leading to a higher yield in pig islet isolation.7,8 However, one study suggested that trypsin could be used to isolate islets from the rat pancreas.9 Soybean trypsin inhibitor (STI) inhibits the activity of trypsin derived from different species.10 To test the effects of trypsin inhibitor on islet yield, we checked islet number, size distribution, and viability in the presence of different concentrations of STI added during collagenase digestion of rat pancreata. MATERIALS AND METHODS Animals Eighty Lewis male rats, weight 200 to 250 g, were anesthetized with intraperitoneal somnotol.
Islet Isolation Pancreatic islets of Langerhans were isolated using the standard technique of collagenase digestion and discontinuous Ficoll gradient purification. Briefly, the common bile duct was cannulated with a PE50 tube, and the pancreas distended with 10 mL chilled University of Wisconsin (UW) solution or Hanks’ balanced salt solution (HBSS; GIBCO), containing glucose (100 mg/dL), penicillin (100 U/mL), and streptomycin (100 g/mL). The pancreas was carefully dissected from surrounding tissues and placed in cooled HBSS (supplemented as above) either for digestion or for exposure to UW solution for 6 to 8 hours cold preservation. Before digestion, each of five pancreata was cut into 1 to 2 mm,3 fragments then digested immediately under five different conditions: group A (n ⫽ 10 for fresh and cold preserved pancreas, respectively): 2 mg/mL type V collagenase (Sigma, ST Louis, Mo) without soybean 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03947-7 488
trypsin inhibitor (STI); Group B (n ⫽ 10 for fresh and cold preserved pancreas, respectively) mixed with 1 mmol/L STI; Group C (n ⫽ 10 for fresh and cold preserved pancreas, respectively) mixed with 2 mmol/L STI; and group D (n ⫽ 10 for fresh and cold preserved pancreas, respectively) mixed with 4 mmol/L STI. After digestion, the islets were filtered through a 220-m nylon screen, then purified by Ficoll density gradient centrifugation.
Cell Count, Size and Morphology The hand-picked islets were counted under a stereomicroscope. After removing 100 islets for the static study and 150 islets for culture, the remaining islets were stained with dithizone, and the mean diameters of all islets smaller than 75 m, were recorded in 50-m increments for calculation of islet equivalent (IEQ) yield according to the international procedure.6
Viability One hundred fifty islets (100 to 200 m in diameter) were put into the CMRL culture medium supplemented with 10 % (vol/vol) fetal calf serum (FCS, Gibco, Grand Island, NY), 10 mmol/L HEPES, 100 U/mL penicillin and 100 g/mL streptomycin for culture (37°C, 5 % CO2, 95 % air for 36 to 48 hrs). For testing viability before and 36 to 48 hours after culture, 25 islets (100 to 200 m in diameter) were hand-picked and incubated in 1.5 mL RPMI medium supplemented with 2 mmol/L L-glutamine, 0.5 % BSA and either 2.8 or 20 mmol/L glucose solution for 2 hours. The insulin concentration of the medium was measured using an ELISA (Boehinger Mannheim).
Statistical Analysis Results are expressed as mean values and standard errors of the mean (X ⫾ S.E). For comparisons among the three groups, differences were analyzed by one-way ANOVA. P ⬍ .05 was considered to be a significant difference. From the Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, Taoyuan, Taiwan and the Department of Surgery-Medicine Institute, University of Alberta, Alberta, Canada. Supported by grants from the Chang-Gung Medical Research Program (CMRP 1090). Address reprint request to Dr. Wen-Tsoung Lu, Division of Endocrinology and Metabolism, Department of Internal Medicine, 5 Fu-Shin St., Kweishan, Taoyuan, Taiwan. © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 488 – 489 (2003)
EFFECT OF TRYPSIN INHIBITOR ON ISLET ISOLATION
RESULTS
Experiments using nonstored pancreata showed the mean (⫾S.E) islet numbers to be 484 ⫾ 23, 456 ⫾ 40, 520 ⫾ 32, 515 ⫾ 44 (P ⫽ .556), and the IEQs were 450.4 ⫾ 27.1, 424.0 ⫾ 34.2, 443.0 ⫾ 28.8, 460.8 ⫾ 31.9 for groups A, B, C, and D, respectively (P ⫽ .859). The insulin responses upon exposure to a 2.8 mM glucose solution were 243 ⫾ 21.2, 206.8 ⫾ 38.3, 210.5 ⫾ 20.7, and 199 ⫾ 11.0 U/L; and to 20 mM glucose solution, 3647.1 ⫾ 245, 3715.5 ⫾ 275.8, 3738 ⫾ 168.5 and 3630 ⫾ 245.4 U/L for Groups A, B, C and D, respectively. In experiments using cold preserved pancreata the mean (⫾S.E) islet numbers were 398 ⫾ 17, 367 ⫾ 23, 401 ⫾ 21 and 391 ⫾ 29 (P ⫽ .713), and the IEQs were 323.3 ⫾ 13.6, 305.7 ⫾ 17.7, 310.4 ⫾ 26.2, and 309.0 ⫾ 23.8 (P ⫽ .938) for the corresponding groups. The insulin responses to 2.8 mmol/L glucose solution were 424.2 ⫾ 55.9, 436.7 ⫾ 38.90, 396.3 ⫾ 233.6, and 409.2 ⫾ 39.1 U/L; and to 20 mmol/L glucose solution, 3875.5 ⫾ 330.0, 4614.5 ⫾ 360.0, 4605 ⫾ 280.0 and 4944.7 ⫾ 346.4 U/L for groups A, B, C, and D, respectively. Compared with a nonstored pancreas, the islet yield from a cold-preserved pancreas was decreased (P ⫽ .02 for mean number; P ⫽ .002 for IEQs). However, the insulin response to 2.8 mmol/L glucose solution was higher among the groups of cold preserved pancreata (P ⬍ .05). Comparing the groups without and with different concentration of STI, there was no significant difference in the total number, IEQs, size distribution, and function when islets were isolated from a fresh or a cold preserved pancreas. CONCLUSION AND DISCUSSION
Using intraductal flushing with UW solution, we obtained functional islets from rat pancreata after 6 to 8 hours of cold storage. Compared with a fresh pancreas, the yield of islet isolated from the cold-preserved pancreas was decreased (P
489
⫽ .02 for mean number; P ⫽ .002 for IEQs). Our data indicate that STI is neither helpful nor harmful to islet yield from either a fresh or a cold-preserved rat pancreas. Another agent—, pefabloc—has been shown to be detrimental on the yield of islets isolated from either a fresh or a cold-preserved rat pancreas.11 However, pefabloc was reported to benefit the isolation of islets from a non-coldpreserved porcine pancreas.7,8 More data are needed to elucidate the optimal trypsin activity during collagenase digestion and to discern whether it is different for islet isolated from various animals due to the presence of other endogenous proteases that might interact with collagenase. More consistent data are needed to support the benefits of exposure to trypsin inhibitor to improve the outcome of digestion of human cadaveric pancreas. REFERENCES 1. Lakey JRT, Rajotte RV, Warnock GL, et al: Transplant 59:689, 1995 2. Benhamou PY, Watt PC, Mullen Y, et al: Transplant 57:1804, 1994 3. White SA, Hughes DP, Contractor HH, et al: J Mol Med 77:79, 1999 4. Johnson PRV, White SA, London NJM: Cell Transplant 5:437, 1996 5. White SA, Djaballah H, Hughes DP, et al: Cell Transplant 8:265, 1999 6. Wolters GHJ, Suylichem PTR, Van Deijnen JHM, et al: Transplant Proc 21:2626, 1989 7. Heiser A, Ulrichs K, Muller-Ruchholtz W: J Clin Lan Anal 8:407, 1994 8. Basir I, van der Burg MP, Scheringa M, et al: Transplant Proc 29:1939, 1997 9. Henriksson C, Bergmark J, Claes G: Eur Surg Res 9:427, 1997 10. Bai RX, Fujimori K, Koja S, et al: Transplant Proc 30:349, 1998 11. Lu WT, Lakey JRT, Juang JH, et al: Transplant Proc 34:2700, 2002
O
BTAINING more islets has limited the progress of human islet transplantation. Multiple factors, including ischemia time, different types of collagenase, distension degree applied to the pancreas, mechanic trauma, and variable digestion might be involved in the low islet recovery rate from a cadaveric pancreas.1–3 Among these factors, the collagenase digestion phase is thought to be the most crucial issue because of the between batch variation.4 In addition to the variable content of exogenous proteases in different collagenase blends, it is more attractive to investigate the variable activity of endogenous proteases released by pancreatic acinar cells during different digestion conditions.5 Wolters et al, described a positive effect (⫹48 %) of a collagenase solution supplemented with 10% bovine serum albumin as a protease inhibitor to increase rat islet yield.6 Two studies reported that pefabloc reduces trypsin activity leading to a higher yield in pig islet isolation.7,8 However, one study suggested that trypsin could be used to isolate islets from the rat pancreas.9 Soybean trypsin inhibitor (STI) inhibits the activity of trypsin derived from different species.10 To test the effects of trypsin inhibitor on islet yield, we checked islet number, size distribution, and viability in the presence of different concentrations of STI added during collagenase digestion of rat pancreata. MATERIALS AND METHODS Animals Eighty Lewis male rats, weight 200 to 250 g, were anesthetized with intraperitoneal somnotol.
Islet Isolation Pancreatic islets of Langerhans were isolated using the standard technique of collagenase digestion and discontinuous Ficoll gradient purification. Briefly, the common bile duct was cannulated with a PE50 tube, and the pancreas distended with 10 mL chilled University of Wisconsin (UW) solution or Hanks’ balanced salt solution (HBSS; GIBCO), containing glucose (100 mg/dL), penicillin (100 U/mL), and streptomycin (100 g/mL). The pancreas was carefully dissected from surrounding tissues and placed in cooled HBSS (supplemented as above) either for digestion or for exposure to UW solution for 6 to 8 hours cold preservation. Before digestion, each of five pancreata was cut into 1 to 2 mm,3 fragments then digested immediately under five different conditions: group A (n ⫽ 10 for fresh and cold preserved pancreas, respectively): 2 mg/mL type V collagenase (Sigma, ST Louis, Mo) without soybean 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03947-7 488
trypsin inhibitor (STI); Group B (n ⫽ 10 for fresh and cold preserved pancreas, respectively) mixed with 1 mmol/L STI; Group C (n ⫽ 10 for fresh and cold preserved pancreas, respectively) mixed with 2 mmol/L STI; and group D (n ⫽ 10 for fresh and cold preserved pancreas, respectively) mixed with 4 mmol/L STI. After digestion, the islets were filtered through a 220-m nylon screen, then purified by Ficoll density gradient centrifugation.
Cell Count, Size and Morphology The hand-picked islets were counted under a stereomicroscope. After removing 100 islets for the static study and 150 islets for culture, the remaining islets were stained with dithizone, and the mean diameters of all islets smaller than 75 m, were recorded in 50-m increments for calculation of islet equivalent (IEQ) yield according to the international procedure.6
Viability One hundred fifty islets (100 to 200 m in diameter) were put into the CMRL culture medium supplemented with 10 % (vol/vol) fetal calf serum (FCS, Gibco, Grand Island, NY), 10 mmol/L HEPES, 100 U/mL penicillin and 100 g/mL streptomycin for culture (37°C, 5 % CO2, 95 % air for 36 to 48 hrs). For testing viability before and 36 to 48 hours after culture, 25 islets (100 to 200 m in diameter) were hand-picked and incubated in 1.5 mL RPMI medium supplemented with 2 mmol/L L-glutamine, 0.5 % BSA and either 2.8 or 20 mmol/L glucose solution for 2 hours. The insulin concentration of the medium was measured using an ELISA (Boehinger Mannheim).
Statistical Analysis Results are expressed as mean values and standard errors of the mean (X ⫾ S.E). For comparisons among the three groups, differences were analyzed by one-way ANOVA. P ⬍ .05 was considered to be a significant difference. From the Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, Taoyuan, Taiwan and the Department of Surgery-Medicine Institute, University of Alberta, Alberta, Canada. Supported by grants from the Chang-Gung Medical Research Program (CMRP 1090). Address reprint request to Dr. Wen-Tsoung Lu, Division of Endocrinology and Metabolism, Department of Internal Medicine, 5 Fu-Shin St., Kweishan, Taoyuan, Taiwan. © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 488 – 489 (2003)
EFFECT OF TRYPSIN INHIBITOR ON ISLET ISOLATION
RESULTS
Experiments using nonstored pancreata showed the mean (⫾S.E) islet numbers to be 484 ⫾ 23, 456 ⫾ 40, 520 ⫾ 32, 515 ⫾ 44 (P ⫽ .556), and the IEQs were 450.4 ⫾ 27.1, 424.0 ⫾ 34.2, 443.0 ⫾ 28.8, 460.8 ⫾ 31.9 for groups A, B, C, and D, respectively (P ⫽ .859). The insulin responses upon exposure to a 2.8 mM glucose solution were 243 ⫾ 21.2, 206.8 ⫾ 38.3, 210.5 ⫾ 20.7, and 199 ⫾ 11.0 U/L; and to 20 mM glucose solution, 3647.1 ⫾ 245, 3715.5 ⫾ 275.8, 3738 ⫾ 168.5 and 3630 ⫾ 245.4 U/L for Groups A, B, C and D, respectively. In experiments using cold preserved pancreata the mean (⫾S.E) islet numbers were 398 ⫾ 17, 367 ⫾ 23, 401 ⫾ 21 and 391 ⫾ 29 (P ⫽ .713), and the IEQs were 323.3 ⫾ 13.6, 305.7 ⫾ 17.7, 310.4 ⫾ 26.2, and 309.0 ⫾ 23.8 (P ⫽ .938) for the corresponding groups. The insulin responses to 2.8 mmol/L glucose solution were 424.2 ⫾ 55.9, 436.7 ⫾ 38.90, 396.3 ⫾ 233.6, and 409.2 ⫾ 39.1 U/L; and to 20 mmol/L glucose solution, 3875.5 ⫾ 330.0, 4614.5 ⫾ 360.0, 4605 ⫾ 280.0 and 4944.7 ⫾ 346.4 U/L for groups A, B, C, and D, respectively. Compared with a nonstored pancreas, the islet yield from a cold-preserved pancreas was decreased (P ⫽ .02 for mean number; P ⫽ .002 for IEQs). However, the insulin response to 2.8 mmol/L glucose solution was higher among the groups of cold preserved pancreata (P ⬍ .05). Comparing the groups without and with different concentration of STI, there was no significant difference in the total number, IEQs, size distribution, and function when islets were isolated from a fresh or a cold preserved pancreas. CONCLUSION AND DISCUSSION
Using intraductal flushing with UW solution, we obtained functional islets from rat pancreata after 6 to 8 hours of cold storage. Compared with a fresh pancreas, the yield of islet isolated from the cold-preserved pancreas was decreased (P
489
⫽ .02 for mean number; P ⫽ .002 for IEQs). Our data indicate that STI is neither helpful nor harmful to islet yield from either a fresh or a cold-preserved rat pancreas. Another agent—, pefabloc—has been shown to be detrimental on the yield of islets isolated from either a fresh or a cold-preserved rat pancreas.11 However, pefabloc was reported to benefit the isolation of islets from a non-coldpreserved porcine pancreas.7,8 More data are needed to elucidate the optimal trypsin activity during collagenase digestion and to discern whether it is different for islet isolated from various animals due to the presence of other endogenous proteases that might interact with collagenase. More consistent data are needed to support the benefits of exposure to trypsin inhibitor to improve the outcome of digestion of human cadaveric pancreas. REFERENCES 1. Lakey JRT, Rajotte RV, Warnock GL, et al: Transplant 59:689, 1995 2. Benhamou PY, Watt PC, Mullen Y, et al: Transplant 57:1804, 1994 3. White SA, Hughes DP, Contractor HH, et al: J Mol Med 77:79, 1999 4. Johnson PRV, White SA, London NJM: Cell Transplant 5:437, 1996 5. White SA, Djaballah H, Hughes DP, et al: Cell Transplant 8:265, 1999 6. Wolters GHJ, Suylichem PTR, Van Deijnen JHM, et al: Transplant Proc 21:2626, 1989 7. Heiser A, Ulrichs K, Muller-Ruchholtz W: J Clin Lan Anal 8:407, 1994 8. Basir I, van der Burg MP, Scheringa M, et al: Transplant Proc 29:1939, 1997 9. Henriksson C, Bergmark J, Claes G: Eur Surg Res 9:427, 1997 10. Bai RX, Fujimori K, Koja S, et al: Transplant Proc 30:349, 1998 11. Lu WT, Lakey JRT, Juang JH, et al: Transplant Proc 34:2700, 2002